Pest Management Science

Pest Manag Sci 58:3337 (online: 2001) DOI: 10.1002/ps.414

Antimicrobial and pesticidal activity of partially puried avonoids of Annona squamosa

Hemlata M Kotkar, Prashant S Mendki, Sangeetha VGS Sadan, Shipra R Jha, Shripad M Upasani and Vijay L Maheshwari*
School of Life Sciences, North Maharashtra University, PB No 80, Jalgaon, India

Abstract: Foliar extracts of Annona squamosa (Family: Annonaceae) were screened for antimicrobial and insecticidal activity against the common microbial infestants of pulses and the stored grain pest pulse beetle, Callosobruchus chinensis (Coleoptera: Bruchidae). Flavonoids isolated from aqueous extracts of A squamosa showed antimicrobial activity against all the common microbial contaminants of pulses and 80% insecticidal activity against C chinensis at a concentration of 0.07 mg ml1. Various physico-chemical tests, chromatographic and spectroscopic studies with partially puried aqueous extract indicated the presence of avonol type avonoids. This may provide a useful beginning for the development of botanical pesticides for post-harvest safeguard of pulses. # 2001 Society of Chemical Industry

Keywords: Annona squamosa; Callosobruchus chinensis; pulses; avonoids; biopesticide

1 INTRODUCTION

In the average south Asian and west African diet, pulses are the major and cost-effective source of proteins. Although India is the largest producer of pulses, it has to import to meet basic demands due to (1) an increasing population, (2) stagnation in production (in 1960 pulses production was 12 and in 1998 it was 14 million tonnes), (3) less production per hectare1 and (4) losses during storage due to infestation by pulse beetle Callosobruchus chinensis L.2 Synthetic pesticides such as pyrethroids, carbamates and organophosphates are known for their rapid knock-down effect, but their incessant use has resulted in several environmental as well as biological problems.3 Currently, emphasis is accorded to products that are biodegradable and eco-friendly to protect grain during storage against insect infestation.4 In this connection, we have explored the use of Calotropis procera R Br, Nerium indicum L and Carica papaya L, in the form of leaf dusts and their foliar extracts, to combat the attack of the stored grain pest, the pulse beetle, C chinensis.5,6 Various extracts of Annona squamosa L are antimicrobial7,8 and its seed oil is larvicidal against Tribolium castaneum (Herbst) and mosquito.9,10 Leaves of A squamosa are known to contain various types of avonoids, some of which can operate as phytoalexins.11 These are mainly involved in the defence mechanisms of plants, and some are known to possess signicant antimicrobial and insecticidal properties.12,13 On the basis of the above facts, an

aqueous leaf extract of A squamosa was examined as a source of avonoids to inhibit the proliferation of micro-organisms and the insect pest C chinensis for the protection of pulses stored under Indian conditions.

2 EXPERIMENTAL METHODS 2.1 Maintenance of microbial cultures

Two species each of Pseudomonas and Bacillus and one each of Cellulomonas and Aspergillus were maintained at room temperature on nutrient agar (pH 7.0 (0.2)) and Czapek Dox agar (pH 5.0 (0.2)), respectively.
2.2 Rearing conditions of Callosobruchus chinensis

Insects were reared under standardised laboratory conditions. Food grade plastic jars (Sunpet, India), containing 50 g of pulses (green gram, Vigna radiata (L) Wilczek) as a nutritional source, were infested with 15 pairs of insects in each jar. The average daily temperature was 30 (2) C and relative humidity was 6070%.14
2.3 Foliar extraction and isolation of avonoids of Annona squamosa

Leaves of A squamosa were collected locally. To prepare foliar extracts, fresh leaves (100 g) were soxhlet-extracted in water (1000 ml) at 95 C. Flavonoids were isolated from this extract by slightly modifying the method described by Agarwal.15 The aqueous extract was concentrated ve-fold in a rotary

* Correspondence to: Vijay L Maheshwari, School of Life Sciences, North Maharashtra University, PB No 80, Jalgaon, India E-mail: vlmaheshwari@hotmail.com Contract/grant sponsor: CM Fund Contract/grant sponsor: CSIR, New Delhi (Received 16 July 2001; accepted 13 August 2001)

# 2001 Society of Chemical Industry. Pest Manag Sci 1526498X/2002/$30.00

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HM Kotkar et al

vacuum evaporator (Buchi, Switzerland) at 5060 C and fractionated with hexane (5 100 ml). To this, lead acetate (10 g litre1; 10 ml) was added to precipitate tannins. The precipitate was discarded and the supernatant diluted with distilled water (1 1), acidied with hydrochloric acid (2 M) and heated on a boiling water bath for 30 min. The precipitate thus formed was dissolved in methanol and used for further experiments as partially puried avonoids.

2.4 Various bioassays of partially puried avonoids

2.5.1 Thin layer chromatography (TLC) and high performance thin layer chromatography (HPTLC) To perform TLC and HPTLC studies, standard silica gel 60 F254 plates (E Merck) were used. A standard solvent system of ethyl acetate formic acid glacial acetic acid water (100 11 11 27 by volume) was employed16 to detect the type of avonoid present in the aqueous leaf extract. For band application on HPTLC plates, Linomat-4 was used (CAMAG, Switzerland). After drying, the plates were observed in a TLC scanner 3 (CAMAG, Switzerland) at 254 and 365 nm.16 2.5.2 Detection of flavonoids The plates were sprayed with methanolic diphenylboric acid b-ethylamino ester (NP; 10 g litre1; 10 ml), followed by ethanolic polyethyleneglycol 4000 (PEG; 50 g litre1; 8 ml) (NP/PEG-4000 reagent) which showed a characteristic avonoid colour immediately or within 15 min at 365 nm.16,17

2.4.1 Plate exposure studies in storage room using mixture of crude flavonoids For these studies, a routine poured plate technique was employed, in which nutrient agar and avonoids were added (pH 7.0 (0.2)). Plates were then exposed in ve different areas of the storage room (right top, left top, centre, right bottom and left bottom) for 20 min. A negative (master) and solvent control were included. The plates were incubated at room temperature for 48 h. Bacterial and fungal colonies were counted at intervals of 24 h. 2.4.2 Micro-aerosol spray studies using crude flavonoids A formulation of 0.07 mg ml1 partially puried avonoids, diluted in distilled water, was sprayed in the storage room using a humidier with microaerosol spray (Ashwini Equipment, Pune). A microbial count was made before and three days after spraying. 2.4.3 Antimicrobial assay The assay was performed with partially puried avonoids (0.07 mg ml1) by the routine borewell technique using King's B medium comprising peptone (20 g litre1), glycerol (10 g litre1), KH2PO4 (1.5 g litre1), MgSO4 (1.5 g litre1) and agar (15 g litre1), and the pH was adjusted to 7.0 ( 0.2) for bacteria and 5.0 ( 0.2) for fungi. 2.4.4 Bioefficacy testing of crude flavonoids against Callosobruchus chinensis To demonstrate pesticidal activity, partially puried avonoids at various suitable concentrations were sprayed onto a Petri plate preloaded with a Whatman No 1 lter paper, and 25 adult C chinensis then added. The plates were kept at room temperature and the effect of partially puried avonoids on the beetles was monitored every hour for 9 h. Each set was run in triplicate and repeated three times in all three seasons.

3 RESULTS AND DISCUSSION

2.5 Characterization of partially puried avonoids

Various physico-chemical tests and preliminary spectroscopic studies described by Agarwal15 were carried out for the characterization of partially puried avonoids.
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Previous studies had revealed that, during storage, moisture content increases as a function of time, favouring microbial attack, which in turn facilitates insect infestation in stored grain.5,14 In addition, bacterial and fungal infestation is known to cause about 7% loss of food and feed stuffs.18 Therefore, before proceeding to a bioefcacy study against C chinensis, the antimicrobial activity of the partially puried avonoids isolated from aqueous leaf extract of A squamosa was screened against the common microbial infestants of stored pulses. Antimicrobial activity of avonoids is attributed to an alteration in membrane structures,12 whereas insecticidal activity is mainly the result of inhibition of vital enzymatic pathways, such as cytochrome-P 450, in which the avonoids specically block steroid hydroxylase involved in the moulting process of insect pests.19 Table 1 presents the results of plate exposure studies carried out in a storage room. The number of colonies observed at all concentrations in the experimental sets were less than in the master and solvent controls, with total arrest of microbial growth observed at 0.07 mg ml1 of avonoids. Similarly, the concentration of avonoids required for 50% inhibition of microorganisms (LC50) was 0.04 mg ml1. On the basis of these results, a water-based microaerosol spray was formulated having an effective avonoid concentration of 0.07 mg ml1. The formulation was found to control the microbial growth effectively for at least the rst 48 h after spraying (Table 2), thus making it a potentially effective spray to control microbial contamination in storage rooms and in general microbiological laboratories. The partially puried mixture of avonoids was also screened for antimicrobial activity by the agar diffusion method against various micro-organisms isolated from infested pulses and storage rooms. Zones of
Pest Manag Sci 58:3337 (online: 2001)

Antimicrobial and pesticidal activity of avonoids

Table 1. Number of microbial colonies at various concentrations of avonoids

Number of colonies at avonoid concentration 0.005 (mg ml Master control Solvent control Experimental SEa SEDa CDa
1

0.01 (mg ml

0.04 (mg ml 1) 28.40 (5.29) 22.00 (5.29) 12.60 (3.79) 2.04 2.89 6.67

0.05 (mg ml 1) 27.00 (10.65) 22.00 (14.19) 12.60 (6.02) 2.69 3.80 8.77

0.06 (mg ml 1) 24.80 (1.92) 24.00 (12.47) 5.60 (3.84) 3.71 5.25 12.11

0.07 (mg ml 1) 25.80 (4.91) 9.00 (4.89) 0.40 (0.89) 1.81 2.27 5.93

21.20 (6.05) 18.20 (11.49) 16.80 (8.89) 3.48 4.92 11.35

28.20 (15.80) 27.60 (10.80) 17.60 (7.79) 4.16 5.89 13.59

a SE = standard error, SED = standard error difference, CD = critical difference (P = 0.05). Statistical analysis was done on Indostat software by ANOVA.

Microbial count a,b (SD) after incubation for 24 h Days after spraying c Before spraying After spraying Day 1 Day 2 Day 3
Table 2. Proles of micro-aerosol spray of avonoids of Annona squamosa at 0.07 mg ml 1 on microbial count
a b

48 h Bacterial count 30.2 (11.6) 12.8 (4.5) 14.0 (6.5) 30.0 (9.3) Fungal count 6.2 (1.1) 2.6 (1.2) 3.0 (1.5) 7.0 (2.3)

Bacterial count 20 (4.5) 5.8 (2.2) 7.8 (4.5) 11.2 (6.4)

Fungal colonies were not visible at 24 h. n = 3. c Day 1 represents exposure of plates in the storage room on the day of spraying.

Table 3. Antimicrobial proles of crude avonoids of Annona squamosa

Micro-organism Pseudomonas 1 Pseudomonas 2 Bacillus 1 Bacillus 2 Cellulomonas sp Aspergillus sp Untreated (master control) Solvent control
a

Zone of inhibition (mm) (SD) a 14.66 (1.24) 14.33 (0.94) 12.66 (1.24) 19.66 (1.24) 15.33 (0.47) 4.33 (1.24) Nil Nil

n = 3.

inhibition against Pseudomonas, Bacillus and Aspergillus species were encouraging, as these are common contaminants of pulses under Indian storage condi-

tions (Table 3).20 Both the Pseudomonas spp were isolated from infested pulses, and Bacillus and Aspergillus from the plate exposure assay in the storage room. Aspergillus is found everywhere under Indian storage conditions; it attacks various proteinase inhibitors of pulses and thereby invites insect attack.6,14 Table 4 shows the insecticidal activity at various concentrations of avonoids. At 0.07 mg ml1 of partially puried avonoids, the mortality of C chinensis was 80% and increased to 100% at 0.09 mg ml1. In our studies, 80% mortality of pulse beetle after 9 h suggest that these avonoids are systemic, rather than contact poisons or anti-feedants. Results of preliminary tests carried out for differentiation and identication, indicated the avonoids to be of the avonol type (Table 5). Flavonoids were further identied by TLC and HPTLC using the

Table 4. Effect of various concentrations of avonoids on Callosobruchus chinensis

Mortality (%) at avonoid concentration 0.01 (mg ml Solvent control Master control Experimental SEa SEDa CDa
a

0.03 (mg ml 1) 5.66 (1.52) 0.00 9.66 (0.57) 0.45 0.63 1.77

0.05 (mg ml 1) 8.00 (1.00) 0.00 41.00 (1.00) 0.57 0.81 2.26

0.07 (mg ml 1) 21.00 (1.00) 0.00 81.33 (1.52) 0.73 1.03 2.87

0.09 (mg ml 1) 29.66 (1.52) 0.00 99 (1.00) 0.45 0.63 1.77

6.00 (1.00) 0.00 9.66 (0.57) 0.45 0.63 1.77

SE = standard error, SED = standard error difference, CD = critical difference (P = 0.05). Statistical analysis was done on Indostat software by ANOVA.

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HM Kotkar et al Test Partially puried avonoids dilute sodium hydroxide Partially puried avonoids concentrated sulfuric acid Partially puried avonoids magnesium ion/hydrochloric acid l max (peaks in absorption spectroscopy) Observation Yellow colour Crimson red colour Red colour 358, 271 nm

Table 5. Preliminary tests for the identication of crude avonoids

Table 6. Rf values of avonoids of Annona squamosa on silica gel 60F254 plate

Probable avonoid a Quercetin-3-O-rutinoside, rutin, o-br Myricetin-3-O-galactoside, o-br Quercetin-3-O-glycoside, isoquercitrin, br-o Quercetin-3,7-dimethyl ether, quercitrin, o
a

Rf b 0.34 0.45 0.53 0.66

Typical colour of avonoids after NP/PEG-4000 reagent treatment on silica gel are indicated as: o = orange, br = brown. b System 1: ethyl acetate formic acid glacial acetic acid water (100 11 11 27 by volume).

specic solvent system mentioned earlier (Table 6).16,17 They were separated on the basis of polarity, which is determined by the ring substituents and the different sugar units attached to the parent molecule.17 The separation of avonol glycosides of A squamosa, on the basis of HPTLC, with uorescence quenching as detection mode under deuterium (254 nm) and mercury lamp (366 nm) is indicated in Figs 1 and 2.17 The separated compounds gave well-dened bands with Rf values ranging from 0.34 to 0.66 with ethyl

acetate formic acid glacial acetic acid water as the mobile phase (Table 6). Overall, four bands of avonoids were observed which showed a typical colour (or uorescence which is structure-dependent) after spraying TLC/HPTLC plates with NP/PEG4000, and Rf values comparable with the standard Rf values.16 On the basis of HPTLC analysis, the prominent peak was myricetin-3-O-galactoside (peak 3; Figs 1 and 2) which is known to inhibit ecdysone 20monooxygenase activity.19 All the above-mentioned avonoids were reported to be potent anti-microbial and insecticidal phytochemicals.12,13,19 Most of these compounds are present in drugs as mono- or diglycosides. The main constituents of avonoid drugs are 2phenyl-g-benzopyrones.16 All these studies conrmed the presence of avonol type of glycosides in the aqueous extract of A squamosa.

4 CONCLUSION

From the above observations it can be concluded that a mixture of partially puried avonoids of A squamosa can be used effectively to minimize the post-harvest losses in pulses due to common microbial contaminants and the pulse beetle, C chinensis, the major

Figure 1. Separation of avonol glycosides from Annona squamosa. a and b indicate application of sample at the rate of 10 ml and 20 ml. Solvent system: ethyl acetate formic acid acetic acid water (100 11 11 27). Stationary phase: Silica gel 60 F254 (0.25 mm, Merck). Developing distance: 7.74 cm. Detection: Absorbance at 254 nm (uorescence quenching in reection mode). Peak identities: (1) quercitrin, (2) isoquercitrin, (3) myricetin-3-O-galactoside and (4) rutin (quercetin-3-Orutinoside) (tentative).

Figure 2. Separation of avonol glycosides from Annona squamosa. a and b indicate application of sample at the rate of 10 ml and 20 ml. Solvent system: ethyl acetate formic acid acetic acid water (100 11 11 27). Stationary phase: silica gel 60F254 (0.25 mm, Merck). Developing distance: 7.74 cm. Detection: absorbance at 366nm (uorescence quenching in reection mode). Peak identities: (1) quercitrin, (2) isoquercitrin, (3) myricetin-3-O-galactoside and (4) rutin (quercetin-3-Orutinoside) (tentative).

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Pest Manag Sci 58:3337 (online: 2001)

Antimicrobial and pesticidal activity of avonoids

stored-grain pest of pulses. To our knowledge, this is the rst report on the isolation and partial characterization of pesticidal avonoids from A squamosa.
ACKNOWLEDGEMENTS

The authors are grateful to Prof RM Kothari, Director, School of Life Sciences for providing laboratory facilities and to Prof SF Patil, Vice-Chancellor, NMU, Jalgaon for the award of CM Fund Fellowship to HMK. Senior Research Fellowship from CSIR, New Delhi to PSM is also gratefully acknowledged. Thanks are due to Mr Dilip Charegaonkar, MD, Anchrom Enterprises (I) Pvt Ltd Mumbai for providing the HPTLC facility.
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